During the previous PPG, we showed that the CD28, CTLA4, and ICOS pathways provide critical second signals for the activation of T cells, isotype switching in B cells, and regulation of autoimmunity. During the tenure of the PPG we identified new co-stimulatory molecules (PD-L1/L2 and TIM-3) and delineated new pathways for T cell activation and expansion. However, it has been difficult to establish which of the many co-stimulatory pathways are most crucial in the induction of human autoimmune diseases. This has fundamentally changed because of the major advances that have been made in the genetic analysis of human autoimmune diseases and of congenic mice. Both of these independent investigations converge on two major loci: one on human chromosome 1p12 which contains CD2, CD48/CD58, CD101 and B7H4, and a second locus on chromosome 2 which contains CD28, CTLA4 and ICOS genes. Within the second locus, CTLA4 has emerged to be the most critical because the isoforms of CTLA4 appear to regulate autoimmunity in both mouse and humans. With these new developments and preliminary data, we have refocused this PPG application to study the co-stimulatory molecules/pathways, which based on the genetic data, appear to be critical for the development of autoimmunity in humans. In the renewal application we are proposing to 1) analyze functions of co-stimulatory molecules (CD2, CD58, B7-H4 and CD101) expressed in 1p12 locus and 2) study the mechanism by which CTLA-4 variants regulate tolerance and autoimmunity. Our studies will investigate human diseases (MS and diabetes) and mouse models of these diseases (EAE and diabetes in the NOD mice). Specifically, Wicker and Todd will analyze the genetic variants in mouse chromosome 3 which correspond to the 1p12 locus and in CTLA4 and CD101 in patients with T1D; Hafler and DeJager will analyze the function of the co-stimulatory molecules identified in the 1p12 locus and CTLA4 in relation to human MS; Kuchroo will generate transgenic mice for CTLA4 variants and analyze their effect on the development of autoimmunity in the mouse models; and Sharpe will test the role of B7-H4 and CD48 (mouse ligand for CD2) in the induction of autoimmunity and tolerance. Insights developed by analyzing the co-stimulatory pathways and genetic variants linked to human and mouse autoimmune diseases will enable us to understand the mechanisms by which allelic variants of co-stimulatory molecules regulate activation of autoreactive T and B cells and induce autoimmune disease.